Methods for the extraction and cultivation of stem cells have long been investigated; see Punzel M et al, The microenvironment of AFT024 cells maintains primitive human hematopoiesis by counteracting contact mediated inhibition of proliferation, CELL COMMUNICATION & ADHESION (2002), 9, 149-159; Gupta P et al. Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparansulfates Requirement for optimal binding interactions of heparinsulfate with early-acting cytokines and matrix proteins, BLOOD (2000) 95, 147-155; Gupta P et. al., Artificial proteoglycan-like molecules containing heparin sulfate enhance the ability of cytokines to maintain human hematopoietic stem cells in vitro, JOURNAL OF INVESTIGATIVE MEDICINE (1995) 43, 342A, & CLINICAL RESEARCH MEETING; SAN DIEGO; Calif.; USA; May 5-8, 1995; Gupta P et. al., Structurally specific heparin sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche, BLOOD (1998) 92, 4641-4651; Lewis I D et al, Umbilical cord blood cells capable of engrafting in primary secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system BLOOD (2001) 97, 3441-3449; Moore K et al., In vitro maintenance of highly purified transplantable hematopoietic stem cells, BLOOD (1991) 89, 4337-4347; Moore K et. al., Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor like repeat motifs, PNAS USA (1997), 94, 4011-4016; Stringer S E et al., Identification of an MIP-lalpha-binding heparin sulfate oligosaccharide that supports long-term in vitro maintenance of human LTC-ICs. BLOOD (2003) 101, 2243-2245).
In Blood (2001) 97, 3441-3449, Lewis I D et al. describe ex vivo cultures with and without contact with stroma cells for the purpose of long-term maintenance and cultivation (expansion) of supposedly pluripotent cells from umbilical cord blood. The stem cells from umbilical cord blood are cultivated in collagen-coated culture dishes, where the stem cells are in fluid contact with AFT024 feeder cells through a membrane. Obviously, the AFT024 feeder cells release an unknown factor effecting the differentiation arrest during the expansion of the stem cells. Moreover, they describe expansion cultures in a uniform stroma-free MV8 medium containing additional N-desulfated O-sulfated heparin. The culture in the stroma-free MV8 medium with the N-desulfated O-sulfated heparin allows a 180-fold expansion of the TNC cells with the propagation of the CD34+ cells or the CFC and LTC-IC expansions, however, being only one- to twofold. In this context, it is remarkable that the expansion of the CD34+ cells in the stroma-free MV8 medium after seven days is only 1.1-fold (±0,2), and only 2.0-fold after 14 days, with a variance of ±2.0 (!!) (see table 2, page 3444), so that an expansion of the CD34+ cells may not be assumed. In addition, stem cells multiplied in this manner only have limited capability for autoregeneration and multilinear differentiation into myeloid and erythroid cells according to an LTC-IC assay. Stem cells multiplied in this manner cannot differentiate into lymphatic cells (NK and NKT cells).
The prior art therefore has the disadvantage that the expansion of the stem and progenitor cells is very small and that no therapeutically applicable therapeutic agents can be manufactured due to the small number of cells. Moreover, the stem and progenitor cells are not multiplied such that they are still able to differentiate into Immunocompetent lymphatic cells afterwards.